Soybean cultivar 0322581

ABSTRACT

A novel soybean cultivar, designated 0322581, is disclosed. The invention relates to the seeds of soybean cultivar 0322581, to the plants of soybean 0322581 and to methods for producing a soybean plant produced by crossing the cultivar 0322581 with itself or another soybean variety. The invention further relates to hybrid soybean seeds and plants produced by crossing the cultivar 0322581 with another soybean cultivar.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a new and distinctive soybeancultivar, designated 0322581. There are numerous steps in thedevelopment of any novel, desirable plant germplasm. Plant breedingbegins with the analysis and definition of problems and weaknesses ofthe current germplasm, the establishment of program goals, and thedefinition of specific breeding objectives. The next step is selectionof germplasm that possess the traits to meet the program goals. The goalis to combine in a single variety an improved combination of desirabletraits from the parental germplasm. These important traits may includehigher seed yield, resistance to diseases and insects, better stems androots, tolerance to drought and heat, and better agronomic quality.

[0002] Choice of breeding or selection methods depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc.). For highly heritable traits, a choice ofsuperior individual plants evaluated at a single location will beeffective, whereas for traits with low heritability, selection should bebased on mean values obtained from replicated evaluations of families ofrelated plants. Popular selection methods commonly include pedigreeselection, modified pedigree selection, mass selection, and recurrentselection.

[0003] The complexity of inheritance influences choice of the breedingmethod. Backcross breeding is used to transfer one or a few favorablegenes for a highly heritable trait into a desirable cultivar. Thisapproach has been used extensively for breeding disease-resistantcultivars. Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

[0004] Each breeding program should include a periodic, objectiveevaluation of the efficiency of the breeding procedure. Evaluationcriteria vary depending on the goal and objectives, but should includegain from selection per year based on comparisons to an appropriatestandard, overall value of the advanced breeding lines, and number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

[0005] Promising advanced breeding lines are thoroughly tested andcompared to appropriate standards in environments representative of thecommercial target area(s) for three or more years. The best lines arecandidates for new commercial cultivars; those still deficient in a fewtraits may be used as parents to produce new populations for furtherselection.

[0006] These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

[0007] A most difficult task is the identification of individuals thatare genetically superior, because for most traits the true genotypicvalue is masked by other confounding plant traits or environmentalfactors. One method of identifying a superior plant is to observe itsperformance relative to other experimental plants and to a widely grownstandard cultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

[0008] The goal of plant breeding is to develop new, unique and superiorsoybean cultivars and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations. The breeder has no direct control at the cellularlevel. Therefore, two breeders will never develop the same line, or evenvery similar lines, having the same soybean traits.

[0009] Each year, the plant breeder selects the germplasm to advance tothe next generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same cultivar twice by using theexact same original parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior new soybean cultivars.

[0010] The development of new soybean cultivars requires the developmentand selection of soybean varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed is produced bymanual crosses between selected male-fertile parents or by using malesterility systems. These hybrids are selected for certain single genetraits such as pod color, flower color, pubescence color or herbicideresistance which indicate that the seed is truly a hybrid. Additionaldata on parental lines, as well as the phenotype of the hybrid,influence the breeder's decision whether to continue with the specifichybrid cross.

[0011] Pedigree breeding and recurrent selection breeding methods areused to develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. The newcultivars are evaluated to determine which have commercial potential.

[0012] Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁'s. Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

[0013] Mass and recurrent selections can be used to improve populationsof either self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

[0014] Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

[0015] The single-seed descent procedure in the strict sense refers toplanting a segregating population, harvesting a sample of one seed perplant, and using the one-seed sample to plant the next generation. Whenthe population has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

[0016] In a multiple-seed procedure, soybean breeders commonly harvestone or more pods from each plant in a population and thresh themtogether to form a bulk. Part of the bulk is used to plant the nextgeneration and part is put in reserve. The procedure has been referredto as modified single-seed descent or the pod-bulk technique.

[0017] The multiple-seed procedure has been used to save labor atharvest. It is considerably faster to thresh pods with a machine than toremove one seed from each by hand for the single-seed procedure. Themultiple-seed procedure also makes it possible to plant the same numberof seeds of a population each generation of inbreeding. Enough seeds areharvested to make up for those plants that did not germinate or produceseed.

[0018] Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

[0019] Proper testing should detect any major faults and establish thelevel of superiority or improvement over current cultivars. In additionto showing superior performance, there must be a demand for a newcultivar that is compatible with industry standards or which creates anew market. The introduction of a new cultivar will incur additionalcosts to the seed producer, the grower, processor and consumer; forspecial advertising and marketing, altered seed and commercialproduction practices, and new product utilization. The testing precedingrelease of a new cultivar should take into consideration research anddevelopment costs as well as technical superiority of the finalcultivar. For seed-propagated cultivars, it must be feasible to produceseed easily and economically.

[0020] Soybean, Glycine max (L), is an important and valuable fieldcrop. Thus, a continuing goal of plant breeders is to develop stable,high yielding soybean cultivars that are agronomically sound. Thereasons for this goal are obviously to maximize the amount of grainproduced on the land used and to supply food for both animals andhumans. To accomplish this goal, the soybean breeder must select anddevelop soybean plants that have the traits that result in superiorcultivars.

SUMMARY OF THE INVENTION

[0021] According to the invention, there is provided a novel soybeancultivar, designated 0322581. This invention thus relates to the seedsof soybean cultivar 0322581, to the plants of soybean 0322581 and tomethods for producing a soybean plant produced by crossing the soybean0322581 with itself or another soybean line, and the creation ofvariants by mutagenesis or transformation of soybean 0322581.

[0022] Thus, any such methods using the soybean variety 0322581 are partof this invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using soybean variety0322581 as a parent are within the scope of this invention.Advantageously, the soybean variety could be used in crosses with other,different, soybean plants to produce first generation (F₁) soybeanhybrid seeds and plants with superior characteristics.

[0023] In another aspect, the present invention provides for single ormultiple gene converted plants of 0322581. The transferred gene(s) maypreferably be a dominant or recessive allele. Preferably, thetransferred gene(s) will confer such traits as herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, male sterility, enhanced nutritional quality, andindustrial usage. The gene may be a naturally occurring soybean gene ora transgene introduced through genetic engineering techniques.

[0024] In another aspect, the present invention provides regenerablecells for use in tissue culture of soybean plant 0322581. The tissueculture will preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing soybeanplant, and of regenerating plants having substantially the same genotypeas the foregoing soybean plant. Preferably, the regenerable cells insuch tissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, podsor stems. Still further, the present invention provides soybean plantsregenerated from the tissue cultures of the invention.

Definitions

[0025] In the description and tables which follow, a number of terms areused. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

[0026] Allele. Allele is any of one or more alternative forms of a gene,all of which alleles relate to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

[0027] Backcrossing. Backcrossing is a process in which a breederrepeatedly crosses hybrid progeny back to one of the parents, forexample, a first generation hybrid F₁ with one of the parental genotypesof the F₁ hybrid.

[0028] Brown Stem Rot. This is a visual disease score from 1 to 9comparing all genotypes in a given test. The score is based on leafsymptoms of yellowing and necrosis caused by brown stem rot. A score of9 indicates no symptoms. Visual scores range to a score of 1 whichindicates severe symptoms of leaf yellowing and necrosis.

[0029] Cotyledon. A cotyledon is a type of seed leaf. The cotyledoncontains the food storage tissues of the seed.

[0030] Embryo. The embryo is the small plant contained within a matureseed.

[0031] Emergence. This score indicates the ability of the seed to emergewhen planted 3″ deep in sand and with a controlled temperature of 25° C.The number of plants that emerge each day are counted. Based on thisdata, each genotype is given a 1 to 9 score based on its rate ofemergence and percent of emergence. A score of 9 indicates an excellentrate and percent of emergence, an intermediate score of 5 indicatesaverage ratings and a 1 score indicates a very poor rate and percent ofemergence.

[0032] Hilum. This refers to the scar left on the seed which marks theplace where the seed was attached to the pod prior to the seed beingharvested.

[0033] Hypocotyl. A hypocotyl is the portion of an embryo or seedlingbetween the cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

[0034] Iron-Deficiency Chlorosis. Plants are scored 1 to 9 based onvisual observations. A score of 9 means no stunting of the plants oryellowing of the leaves and a score of 1 indicates the plants are deador dying caused by iron-deficiency chlorosis, a score of 5 means plantshave intermediate health with some leaf yellowing.

[0035] Lodging Resistance. Lodging is rated on a scale of 1 to 9. Ascore of 9 indicates erect plants. A score of 5 indicates plants areleaning at a 45° angle in relation to the ground and a score of 1indicates plants are laying on the ground.

[0036] Maturity Date. Plants are considered mature when 95% of the podshave reached their mature color. The number of days are eithercalculated from August 31 or from the planting date.

[0037] Maturity Group. This refers to an agreed-on industry division ofgroups of varieties, based on zones in which they are adapted primarilyaccording to day length or latitude. They consist of very long daylength varieties (Groups 000, 00, 0), and extend to very short daylength varieties (Groups VII, VIII, IX, X).

[0038] Relative Maturity (RM). The term relative maturity is a numericalvalue that is assigned to a soybean variety based on comparisons withthe maturity values of other varieties. The number preceding the decimalpoint in the RM refers to the maturity group. The number following thedecimal point refers to the relative earliness or lateness within eachmaturity group. For example, a 3.0 is an early group III variety, whilea 3.9 is a late group III variety.

[0039] Oil or oil percent. Soybean seeds contain a considerable amountof oil. Oil is measured by NIR spectrophotometry, and is reported on anas is percentage basis.

[0040] Oleic Acid Percent. Oleic acid is one of the five most abundantfatty acids in soybean seeds. It is measured by gas chromatography andis reported as a percent of the total oil content.

[0041] Palmitic Acid Percent. Palmitic acid is one of the five mostabundant fatty acids in soybean seeds. It is measured by gaschromatography and is reported as a percent of the total oil content.

[0042] Phytophthora Tolerance. Tolerance to Phytophthora root rot israted on a scale of 1 to 9, with a score of 9 being the best or highesttolerance ranging down to a score of 1 which indicates the plants haveno tolerance to Phytophthora.

[0043] Phenotypic Score. The Phenotypic Score is a visual rating ofgeneral appearance of the variety. All visual traits are considered inthe score including healthiness, standability, appearance and freedom ofdisease. Ratings are scored from 1 being poor to 9 being excellent.

[0044] Plant Height. Plant height is taken from the top of soil to topnode of the plant and is measured in centimeters.

[0045] Pod. This refers to the fruit of a soybean plant. It consists ofthe hull or shell (pericarp) and the soybean seeds.

[0046] Protein Percent. Soybean seeds contain a considerable amount ofprotein. Protein is generally measured by NIR spectrophotometry, and isreported on an as is percentage basis.

[0047] Pubescence. This refers to a covering of very fine hairs closelyarranged on the leaves, stems and pods of the soybean plant.

[0048] Quantitative Trait Loci (QTL). Quantitative trait loci (QTL)refer to genetic loci that control to some degree numericallyrepresentable traits that are usually continuously distributed.

[0049] Regeneration. Regeneration refers to the development of a plantfrom tissue culture.

[0050] Seed Protein Peroxidase Activity. Seed protein peroxidaseactivity is defined as a chemical taxonomic technique to separatecultivars based on the presence or absence of the peroxidase enzyme inthe seed coat. There are two types of soybean cultivars, those havinghigh peroxidase activity (dark red color) and those having lowperoxidase activity (no color).

[0051] Seed Yield (Bushels/Acre). The yield in bushels/acre is theactual yield of the grain at harvest.

[0052] Seeds per Pound. Soybean seeds vary in seed size, therefore, thenumber of seeds required to make up one pound also varies. This affectsthe pounds of seed required to plant a given area, and can also impactend uses.

[0053] Shattering. The amount of pod dehiscence prior to harvest. Poddehiscence involves seeds falling from the pods to the soil. This is avisual score from 1 to 9 comparing all genotypes within a given test. Ascore of 9 means pods have not opened and no seeds have fallen out. Ascore of 5 indicates approximately 50% of the pods have opened, withseeds falling to the ground and a score of 1 indicates 100% of the podsare opened.

[0054] Single Gene Converted (Conversion). Single gene converted(conversion) plant refers to plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of a variety arerecovered in addition to the single gene transferred into the varietyvia the backcrossing technique or via genetic engineering.

DETAILED DESCRIPTION OF THE INVENTION

[0055] 0322581 is an early maturity group 0 variety with resistance toRoundup™ herbicide conferring tolerance to glyphosate herbicides.0322581 has very high yield potential when compared to lines of similarmaturity and has excellent agronomic characteristics including lodgingresistance.

[0056] Some of the criteria used to select in various generationsinclude: seed yield, lodging resistance, emergence, disease tolerance,maturity, late season plant intactness, plant height and shatteringresistance.

[0057] The cultivar has shown uniformity and stability, as described inthe following variety description information. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity.

[0058] Soybean cultivar 0322581 has the following morphologic and othercharacteristics (based primarily on data collected at Adel, Iowa).

Variety Description Information

[0059] Seed Coat Color: Yellow

[0060] Seed Coat Luster: Shiny

[0061] Hilum Color: (Mature Seed)—Brown

[0062] Cotyledon Color (Mature Seed): Yellow

[0063] Leaflet Shape: Ovate

[0064] Flower Color: Purple

[0065] Pod color: Tan

[0066] Plant Pubescence Color: Tawny

[0067] Plant Habit: Indeterminate

[0068] Maturity Group: 0

[0069] Relative Maturity: 0.3

[0070] Plant Lodging Score: 9

[0071] Leaflet size: Medium

[0072] Plant Height (cm): 71

[0073] Seed Content: Protein: 36.8%; Oil: 18.1%

[0074] Seed Size (G/100 seeds): 14.4

[0075] Physiological Responses: Roundup Ready™ Herbicide: Resistant

[0076] This invention is also directed to methods for producing asoybean plant by crossing a first parent soybean plant with a secondparent soybean plant, wherein the first or second soybean plant is thesoybean plant from the line 0322581. Further, both first and secondparent soybean plants may be from the cultivar 0322581. Therefore, anymethods using the cultivar 0322581 are part of this invention: selfing,backcrosses, hybrid breeding, and crosses to populations. Any plantsproduced using cultivar 0322581 as a parent are within the scope of thisinvention.

[0077] Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using the microprojectile media delivery with the biolisticdevice Agrobacterium-medicated transformation. Transformant plantsobtained with the protoplasm of the invention are intended to be withinthe scope of this invention.

[0078] The cultivar 0322581 is similar to 40064423. While similar to40064423 there are numerous differences including: 0322581 has the genefor resistance to the Roundup™ herbicides and 40064423 does not containthis gene.

Further Embodiments of the Invention

[0079] With the advent of molecular biological techniques that haveallowed the isolation and characterization of genes that encode specificprotein products, scientists in the field of plant biology developed astrong interest in engineering the genome of plants to contain andexpress foreign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed variety or line.

[0080] Plant transformation involves the construction of an expressionvector which will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed soybean plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the soybean plant(s).

[0081] Expression Vectors for Soybean Transformation: MarkerGenes—Expression vectors include at least one genetic marker, operablylinked to a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

[0082] One commonly used selectable marker gene for plant transformationis the neomycin phosphotransferase II (nptII) under the control of plantregulatory signals confers resistance to kanamycin. Fraley et al., Proc.Natl. Acad. Sci. U.S.A., 80:4803 (1983). Another commonly usedselectable marker gene is the hygromycin phosphotransferase gene whichconfers resistance to the antibiotic hygromycin. Vanden Elzen et al.,Plant Mol. Biol., 5:299 (1985).

[0083] Additional selectable marker genes of bacterial origin thatconfer resistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990< Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

[0084] Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

[0085] Another class of marker genes for plant transformation requirescreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS,β-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984).

[0086] Recently, in vivo methods for visualizing GUS activity that donot require destruction of plant tissue have been made available.Molecular Probes publication 2908, Imagene Green™, p. 1-4 (1993) andNaleway et al., J. Cell Biol. 115:151a (1991). However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

[0087] More recently, a gene encoding Green Fluorescent Protein (GFP)has been utilized as a marker for gene expression in prokaryotic andeukaryotic cells. Chalfie et al., Science 263:802 (1994). GFP andmutants of GFP may be used as screenable markers.

[0088] Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

[0089] As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which intitiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

[0090] A. Inducible Promoters—An inducible promoter is operably linkedto a gene for expression in soybean. Optionally, the inducible promoteris operably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in soybean. With aninducible promoter the rate of transcription increases in response to aninducing agent.

[0091] Any inducible promoter can be used in the instant invention. SeeWard et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Mett et al., PNAS 90:4567-4571 (1993)); ln2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

[0092] B. Constitutive Promoters—A constitutive promoter is operablylinked to a gene for expression in soybean or the constitutive promoteris operably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in soybean.

[0093] Many different constitutive promoters can be utilized in theinstant invention. Exemplary constitutive promoters include, but are notlimited to, the promoters from plant viruses such as the 35S promoterfrom CaMV (Odell et al., Nature 313:810-812 (1985) and the promotersfrom such genes as rice actin (McElroy et al., Plant Cell 2:163-171(1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632(1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU(Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten etal., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al.,Mol Gen. Genetics 231:276-285 (1992) and Atanassova et al., PlantJournal 2 (3): 291-300 (1992)).

[0094] The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napusALS3 structural gene (or a nucleotide sequence similarity to saidXba1/NcoI fragment), represents a particularly useful constitutivepromoter. See PCT application WO96/30530.

[0095] C. Tissue-specific or Tissue-preferred Promoters—Atissue-specific promoter is operably linked to a gene for expression insoybean. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in soybean. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

[0096] Any tissue-specific or tissue-preferred promoter can be utilizedin the instant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter—such as that from the phaseolin gene (Murai et al., Science23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci.U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promotersuch as that from cab or rubisco (Simpson et al., EMBO J.4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); ananther-specific promoter such as that from LAT52 (Twell et al., Mol.Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such asthat from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993))or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993).

[0097] Signal Sequences for Targeting Proteins to SubcellularCompartments

[0098] Transport of protein produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall or mitochondroin or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine, during protein synthesis andprocessing, where the encoded protein is ultimately compartmentalized.

[0099] The presence of a signal sequence directs a polypeptide to eitheran intracellular organelle or subcellular compartment or for secretionto the apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

[0100] Foreign Protein Genes and Agronomic Genes—With transgenic plantsaccording to the present invention, a foreign protein can be produced incommercial quantities. Thus, techniques for the selection andpropagation of transformed plants, which are well understood in the art,yield a plurality of transgenic plants which are harvested in aconventional manner, and a foreign protein then can be extracted from atissue of interest or from total biomass. Protein extraction from plantbiomass can be accomplished by known methods which are discussed, forexample, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).

[0101] According to a preferred embodiment, the transgenic plantprovided for commercial production of foreign protein is a soybeanplant. In another preferred embodiment, the biomass of interest is seed.For the relatively small number of transgenic plants that show higherlevels of expression, a genetic map can be generated, primarily viaconventional RFLP, PCR and SSR analysis, which identifies theapproximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see Glick and Thompson, Methodsin Plant Molecular Biology and Biotechnology CRC Press, Boca Raton269:284 (1993). Map information concerning chromosomal location isuseful for proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

[0102] Likewise, by means of the present invention, agronomic genes canbe expressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

[0103] 1. Genes That Confer Resistance to Pests or Disease and ThatEncode:

[0104] A. Plant disease resistance genes. Plant defenses are oftenactivated by specific interaction between the product of a diseaseresistance gene (R) in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen. A plant variety can betransformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.,Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistanceto Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomatoPto gene for resistance to Pseudomonas syringae pv. Tomato encoddes aprotein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2gene for resistance to Pseudomonas syringae).

[0105] B. A gene conferring resistance to a pest, such as soybean cystnematode. See e.g., PCT Application WO96/30517; PCT ApplicationWO93/19181.

[0106] C. A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δendotoxin genescan be purchased from American Type Culture Collection, Manassas, Va.,for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

[0107] D. A lectin. See, for example, the disclose by Van Damme et al.,Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

[0108] E. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

[0109] F. An enzyme inhibitor, for example, a protease or proteinaseinhibitor or an amylase inhibitor. See, for example, Abe et al., J.Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteineproteinase inhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993)(nucleotide sequence of cDNA encoding tobacco proteinase inhibitor 1),Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotidesequence of Streptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat.No. 5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

[0110] G. An insect-specific hormone or pheromone such as an ecdysteroidand juvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

[0111] H. An insect-specific peptide or neuropeptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of Regan, J. Biol. Chem. 269:9 (1994) (expressioncloning yields DNA coding for insect diuretic hormone receptor), andPratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989) (anallostatin is identified in Diploptera puntata). See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

[0112] I. An insect-specific venom produced in nature by a snake, awasp, etc. For example, see Pang et al., Gene 116:165 (1992), fordisclosure of heterologous expression in plants of a gene coding for ascorpion insectotoxic peptide.

[0113] J. An enzyme responsible for a hyperaccumulation of a monterpene,a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

[0114] K. An enzyme involved in the modification, including theost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

[0115] L. A molecule that stimulates signal transduction. For example,see the disclosure by Botella et al., Plant Molec. Biol. 24:757 (1994),of nucleotide sequences for mung bean calmodulin cDNA clones, and Griesset al., Plant Physiol. 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

[0116] M. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

[0117] N. A membrane permease, a channel former or a channel blocker.For example, see the disclosure of Jaynes et al., Plant Sci 89:43(1993), of heterologous expression of a cecropin-β, lytic peptide analogto render transgenic tobacco plants resistant to Pseudomonassolanacearum.

[0118] O. A viral-invasive protein or a complex toxin derived therefrom.For example, the accumulation of viral coat proteins in transformedplant cells imparts resistance to viral infection and/or diseasedevelopment effected by the virus from which the coat protein gene isderived, as well as by related viruses. See Beachy et al., Ann. rev.Phytopathol. 28:451 (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaidc virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

[0119] P. An insect-specific antibody or an immunotoxin derivedtherefrom. Thus, an antibody targeted to a critical metabolic functionin the insect gut would inactivate an affected enzyme, killing theinsect. Cf. Taylor et al., Abstract #497, Seventh Int’l Symposium onMolecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

[0120] Q. A virus-specific antibody. See, for example, Tavladoraki etal., Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

[0121] R. A developmental-arrestive protein produced in nature by apathogen or a parasite. Thus, fungal endo α-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-α-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10:1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2:367 (1992).

[0122] S. A development-arrestive protein produced in nature by a plant.For example, Logemann et al., Bioi/Technology 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivting genehave an increased resistance to fungal disease.

[0123] 2. Genes That Confer Resistance to a Herbicide, For Example:

[0124] A. A herbicide that inhibits the growing point or meristem, suchas an imidazalinone or a sulfonylurea. Exemplary genes in this categorycode for mutant ALS and AHAS enzyme as described, for example, by Lee etal., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

[0125] B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy proprionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

[0126] C. A herbicide that inhibits photosynthesis, such as a triazine(psbA and gs+ genes) and a benzonitrile (nitrilase gene). Przibila etal., Plant Cell 3:169 (1991), describe the transformation ofChlamydomonas with plasmids encoding mutant psbA genes. Nucleotidesequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648to Stalker, and DNA molecules containing these genes are available underATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression ofDNA coding for a glutathione S-transferase is described by Hayes et al.,Biochem. J. 285:173 (1992).

[0127] 3. Genes That Confer or Contribute to a Value-Added Trait, Suchas:

[0128] A. Modified fatty acid metabolism, for example, by transforming aplant with an antisense gene of stearoyl-ACP desaturase to increasestearic acid content of the plant. See Knultzon et al., Proc. Natl.Acad. Sci. U.S.A. 89:2624 (1992).

[0129] B. Decreased phytate content—1) Introduction of aphytase-encoding gene would enhance breakdown of phytate, adding morefree phosphate to the transformed plant. For example, see VanHartingsveldt et al., Gene 127:87 (1993), for a disclosure of thenucleotide sequence of an Aspergillus niger phytase gene. 2) A genecould be introduced that reduced phytate content. In maize, this, forexample, could be accomplished, by cloning and then reintroducing DNAassociated with the single allele which is responsible for maize mutantscharacterized by low levels of phytic acid. See Raboy et al., Maydica35:383 (1990).

[0130] C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Sogaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

[0131] Methods for Soybean Transformation—Numerous methods for planttransformation have been developed, including biological and physical,plant transformation protocols. See, for example, Miki et al.,“Procedures for Introducing Foreign DNA into Plants” in Methods in PlantMolecular Biology and Biotechnology, Glick B. R. and Thompson, J. E.Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

[0132] A. Agrobacterium-mediated Transformation—One method forintroducing an expression vector into plants is based on the naturaltransformation system of Agrobacterium. See, for example, Horsch et al.,Science 227:1229 (1985). A. tumefaciens and A. rhizogenes are plantpathogenic soil bacteria which genetically transform plant cells. The Tiand Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carrygenes responsible for genetic transformation of the plant. See, forexample, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

[0133] B. Direct Gene Transfer—Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation wherein DNA is carried on the surface of microprojectilesmeasuring 1 to 4 μm. The expression vector is introduced into planttissues with a biolistic device that accelerates the microprojectiles tospeeds of 300 to 600 m/s which is sufficient to penetrate plant cellwalls and membranes. Sanford et al., Part. Sci. Technol. 5:27 (1987),Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206(1990), Klein et al., Biotechnology 10:268 (1992). See also U.S. Pat.No. 5,015,580 (Christou, et al.), issued May 14, 1991; U.S. Pat. No.5,322,783 (Tomes, et al), issued Jun. 21, 1994.

[0134] Another method for physical delivery of DNA to plants issonication of target cells. Zhang et al., Bio/Technology 9:996 (1991).Alternatively, liposome or spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes et al., EMBO J.,4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-omithine have also been reported. Hain etal., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant CellPhysiol. 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues have also been described. Donn et al., In Abstracts of VIIthInternational Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spenceret al., Plant Mol. Biol. 24:51-61 (1994).

[0135] Following transformation of soybean target tissues, expression ofthe above-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

[0136] The foregoing methods for transformation would typically be usedfor producing a transgenic variety. The transgenic variety could then becrossed, with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular soybean line using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene. As usedherein, “crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

[0137] Tissue Culture of Soybeans—When the term soybean plant is used inthe context of the present invention, this also includes any single geneconversions of that variety. The term single gene converted plant asused herein refers to those soybean plants which are developed by aplant breeding technique called backcrossing wherein essentially all ofthe desired morphological and physiological characteristics of a varietyare recovered in addition to the single gene transferred into thevariety via the backcrossing technique. Backcrossing methods can be usedwith the present invention to improve or introduce a characteristic intothe variety. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent. The parentalsoybean plant which contributes the gene for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental soybean plant towhich the gene or genes from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol (Poehiman & Sleper, 1994; Fehr, 1987). In atypical backcross protocol, the original variety of interest (recurrentparent) is crossed to a second variety (nonrecurrent parent) thatcarries the single gene of interest to be transferred. The resultingprogeny from this cross are then crossed again to the recurrent parentand the process is repeated until a soybean plant is obtained whereinessentially all of the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred gene from the nonrecurrentparent.

[0138] The selection of a suitable recurrent parent is an important stepfor a successful backcrossing procedure. The goal of a backcrossprotocol is to alter or substitute a single trait or characteristic inthe original variety. To accomplish this, a single gene of the recurrentvariety is modified or substituted with the desired gene from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original variety. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross, one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

[0139] Many single gene traits have been identified that are notregularly selected for in the development of a new variety but that canbe improved by backcrossing techniques. Single gene traits may or maynot be transgenic, examples of these traits include but are not limitedto, male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185, 5,973,234 and 5,977,445, the disclosures of which arespecifically hereby incorporated by reference.

[0140] Further reproduction of the variety can occur by tissue cultureand regeneration. Tissue culture of various tissues of soybeans andregeneration of plants therefrom is well know and widely published. Forexample, reference may be had to Komatsuda, T. et al., “Genotype XSucrose Interactions for Somatic Embryogenesis in Soybean,” Crop Sci.31:333-337 (1991); Stephens, P. A., et al., “Agronomic Evaluation ofTissue-Culture-Derived Soybean Plants,” Theor. Appl. Genet. (1991)82:633-635; Komatsuda, T. et al., “Maturation and Germination of SomaticEmbryos as Affected by Sucrose and Plant Growth Regulators in SoybeansGlycine gracilis Skvortz and Glycine max (L.) Merr.” Plant Cell, Tissueand Organ Culture, 28:103-113 (1992); Dhir, S. et al., “Regeneration ofFertile Plants from Protoplasts of Soybean (Glycine max L. Merr.);Genotypic Differences in Culture Response,” Plant Cell Reports (1992)11:285-289; Pandey, P. et al., “Plant Regeneration from Leaf andHypocotyl Explants of Glycine-wightii (W. and A.) VERDC. var.longicauda,” Japan J. Breed. 42:1-5 (1992); and Shefty, K., et al.,“Stimulation of In Vitro Shoot Organogenesis in Glycine max (Merrill.)by Allantoin and Amides,” Plant Science 81:245-251 (1992); as well asU.S. Pat. No. 5,024,944 issued Jun. 18, 1991 to Collins et al., and U.S.Pat. No. 5,008,200 issued Apr. 16, 1991 to Ranch et al., the disclosuresof which are hereby incorporated herein in their entirety by reference.Thus, another aspect of this invention is to provide cells which upongrowth and differentiation produce soybean plants having thephysiological and morphological characteristics of soybean variety0322581.

[0141] As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and 5,977,445,described certain techniques, the disclosures of which are incorporatedherein by reference.

[0142] This invention also is directed to methods for producing asoybean plant by crossing a first parent soybean plant with a secondparent soybean plant wherein the first or second parent soybean plant isa soybean plant of the variety 0322581. Further, both first and secondparent soybean plants can come from the soybean variety 0322581. Thus,any such methods using the soybean variety 0322581 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using soybean variety0322581 as a parent are within the scope of this invention, includingthose developed from varieties derived from soybean variety 0322581.Advantageously, the soybean variety could be used in crosses with other,different, soybean plants to produce first generation (F₁) soybeanhybrid seeds and plants with superior characteristics. The variety ofthe invention can also be used for transformation where exogenous genesare introduced and expressed by the variety of the invention. Geneticvariants created eitherthrough traditional breeding methods usingvariety 0322581 or through transformation of 0322581 by any of a numberof protocols known to those of skill in the art are intended to bewithin the scope of this invention.

[0143] As used herein, the term plant includes plant cells, plantprotoplasts, plant cell tissue cultures from which soybean plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,pods, leaves, roots, root tips, anthers, and the like.

Industrial Uses

[0144] The seed of soybean variety 0322581, the plant produced from theseed, the hybrid soybean plant produced from the crossing of the varietywith any other soybean plant, hybrid seed, and various parts of thehybrid soybean plant can be utilized for human food, livestock feed, andas a raw material in industry.

[0145] The soybean is the world's leading source of vegetable oil andprotein meal. The oil extracted from soybeans is used for cooking oil,margarine, and salad dressings. Soybean oil is composed of saturated,monounsaturated and polyunsaturated fatty acids. It has a typicalcomposition of 11% palmitic, 4% stearic, 25% oleic, 50% linoleic and 9%linolenic fatty acid content (“Economic Implications of Modified SoybeanTraits Summary Report”, Iowa Soybean Promotion Board and AmericanSoybean Association Special Report 92S, May 1990). Changes in fatty acidcomposition for improved oxidative stability and nutrition areconstantly sought after. Industrial uses of soybean oil which issubjected to further processing include ingredients for paints,plastics, fibers, detergents, cosmetics and lubricants. Soybean oil maybe split, inter-esterified, sulfurized, epoxidized, polymerized,ethoxylated, or cleaved. Designing and producing soybean oil derivativeswith improved functionality, oliochemistry, is a rapidly growing field.The typical mixture of triglycerides is usually split and separated intopure fatty acids, which are then combined with petroleum-derivedalcohols or acids, nitrogen, sulfonates, chlorine, or with fattyalcohols derived from fats and oils.

[0146] Soybean is also used as a food source for both animals andhumans. Soybean is widely used as a source of protein for animal feedsfor poultry, swine and cattle. During processing of whole soybeans, thefibrous hull is removed and the oil is extracted. The remaining soybeanmeal is a combination of carbohydrates and approximately 50% protein.

[0147] For human consumption soybean meal is made into soybean flourwhich is processed to protein concentrates used for meat extenders orspecialty pet foods. Production of edible protein ingredients fromsoybean offers a healthy, less expensive replacement for animal proteinin meats as well as dairy-type products.

[0148] As shown in Table 1, soybean cultivar 0322581 yields higher thanfour commercial varieties with the increase over CSR0070 beingsignificant at the 0.01 level of probability and the increase overP90B31 being significant at the 0.05 level.

Tables

[0149] In Table 1 that follows, the traits and characteristics ofsoybean cultivar 0322581 are compared to several competing varieties ofcommercial soybeans of similar maturity. In the tables, column 1 showsthe comparison number; column 2 is the year of the test; columns 3 and 4give the number of locations and number of observations, respectively.Column 5 indicates the genotype and column 6 shows the mean yield inbushels per acre. Column 7 presents the t value and columns 8 and 9present the critical t values at the 0.05% and 0.01 % levels ofsignificance, respectively. TABLE 1 PAIRED COMPARISONS Comp # of # ofMean t Critical Critical # Year Loc. Obs. Genotype Yield Value t@.05t@.01 1 2001 11 32 0322581 40.8 4.10** 1.70 2.45 CS R0070 37.4 2 2001 1132 0322581 40.8 1.30 1.70 2.45 CS R0090 39.5 3 2001 11 32 0322581 40.81.38 1.70 2.45 FLS2601R 39.2 4 2001 11 32 0322581 40.8 2.09* 1.70 2.45P90B31 38.3

Deposit Information

[0150] A deposit of the soybean seed of this invention is maintained byStine Seed Farm, Inc., 2225 Laredo Trail, Adel, Iowa 50003. Access tothis deposit will be available during the pendency of this applicationto persons determined by the Commissioner of Patents and Trademarks tobe entitled thereto under 37 CFR 1.14 and 35 USC 122. Upon allowance ofany claims in this application, all restrictions on the availability tothe public of the variety will be irrevocably removed by affordingaccess to a deposit of at least 2,500 seeds of the same variety with theAmerican Type Culture Collection, Manassas, Va.

[0151] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somoclonal variants, variant individuals selected from large populationsof the plants of the instant variety and the like may be practicedwithin the scope of the invention, as limited only by the scope of theappended claims.

What is claimed is:
 1. Seed of a soybean variety designated 0322581,representative seed having been deposited under ATCC Accession No.______.
 2. A soybean plant, or parts thereof, produced by growing theseed of claim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule of theplant of claim
 2. 5. A soybean plant, or parts thereof, having all ofthe physiological and morphological characteristics of the soybean plantof claim
 2. 6. A tissue culture of regenerable cells from the plant ofclaim
 2. 7. A tissue culture according to claim 6, wherein a cell orprotoplast of the tissue culture is derived from a tissue selected fromthe group consisting of: leaves, pollen, embryos, cotyledon, hypocotyl,meristematic cells, roots, root tips, anthers, flowers, seeds, stems andpods.
 8. A soybean plant regenerated from the tissue culture of claim 6,wherein the regenerated plant is capable of expressing all of themorphological and physiological characteristics of soybean cultivar0322581, representative seed of said soybean cultivar 0322581 havingbeen deposited under ATCC Accession No. ______.
 9. A soybean plant withall of the physiological and morphological characteristics of soybeanvariety 0322581, wherein said soybean plant is produced by a tissueculture process using the soybean plant of claim 5 as the startingmaterial for such a process.
 10. A method for producing a hybrid soybeanseed comprising crossing a first parent soybean plant with a secondparent soybean plant and harvesting the resultant hybrid soybean seed,wherein said first parent soybean plant or said second parent soybeanplant is the soybean plant of claim
 2. 11. A hybrid soybean seedproduced by the method of claim
 10. 12. A hybrid soybean plant, or partsthereof, produced by growing said hybrid soybean seed of claim
 11. 13. Asoybean seed produced by growing said hybrid soybean plant of claim 12and harvesting the resultant seed.
 14. A method for producing a soybeanvariety 0322581-derived soybean plant, comprising: a) crossing soybeanvariety 0322581, representative samples of said variety having beendeposited under ATCC accession number ______, with a second soybeanplant to yield progeny soybean seed; and b) growing said progeny soybeanseed, under plant growth conditions, to yield said soybean variety0322581-derived soybean plant.
 15. A soybean variety 0322581-derivedsoybean plant, or parts thereof, produced by the method of claim 14,said 0322581-derived soybean plant expressing a combination of at leasttwo 0322581 traits selected from the group consisting of: a relativematurity of approximately 0.08 to 0.8, excellent yield, above averagelodging resistance, and above average general appearance.
 16. A methodfor producing a soybean variety 0322581-derived soybean plant,comprising: a) crossing soybean variety 0322581, representative samplesof said variety having been deposited under ATCC accession number______, with a second soybean plant to yield progeny soybean seed; andb) growing said progeny soybean seed, under plant growth conditions, toyield said soybean variety 0322581-derived soybean plant. c) crossingsaid soybean variety 0322581-derived soybean plant with itself oranother soybean plant to yield additional soybean variety0322581-derived progeny soybean seed; d) growing said progeny soybeanseed of step (a) under plant growth conditions, to yield additionalsoybean variety 0322581-derived soybean plants; and e) repeating thecrossing and growing steps of (a) and (b) from 0 to 7 times to generatefurther soybean variety 0322581-derived soybean plants.
 17. A soybeanvariety 0322581-derived soybean plant, or parts thereof, produced by themethod of claim 16, said 0322581 -derived soybean plant expressing acombination of at least two 0322581 traits selected from the groupconsisting of: a relative maturity of approximately 0.08 to 0.8,excellent yield, above average lodging resistance, and above averagegeneral appearance.
 18. The soybean plant, or parts thereof, of claim 5,wherein the plant or parts thereof have been transformed so that itsgenetic material contains a transgene operably linked to a regulatoryelement and wherein said transgene is selected from the group consistingof: herbicide resistance, insect resistance and disease resistance. 19.A method for producing a soybean plant that contains in its geneticmaterial a transgene, comprising crossing the soybean plant of claim 18with either a second plant of another soybean variety, or anon-transformed soybean plant of the soybean variety 0322581, so thatthe genetic material of the progeny that result from the cross containsa transgene operably linked to a regulatory element.
 20. Soybean plants,or parts thereof, produced by the method of claim
 19. 21. A soybeanplant, or parts thereof, wherein at least one ancestor of said soybeanplant is the soybean plant of claim 2, said soybean plant expressing acombination of at least two 0322581 traits selected from the groupconsisting of: a relative maturity of approximately 0.08 to 0.8,excellent yield, above average lodging resistance, and above averagegeneral appearance.